Exploring ITM2A as a new potential target for brain delivery.

BACKGROUND: Integral membrane protein 2A (ITM2A) is a transmembrane protein expressed in a variety of tissues; little is known about its function, particularly in the brain. ITM2A was found to be highly enriched in human brain versus peripheral endothelial cells by transcriptomic and proteomic studies conducted within the European Collaboration on the Optimization of Macromolecular Pharmaceutical (COMPACT) Innovative Medicines Initiative (IMI) consortium. Here, we report the work that was undertaken to determine whether ITM2A could represent a potential target for delivering drugs to the brain. METHODS: A series of ITM2A constructs, cell lines and specific anti-human and mouse ITM2A antibodies were generated. Binding and internalization studies in Human Embryonic Kidney 293 (HEK293) cells overexpressing ITM2A and in brain microvascular endothelial cells from mouse and non-human primate (NHP) were performed with these tools. The best ITM2A antibody was evaluated in an in vitro human blood brain barrier (BBB) model and in an in vivo mouse pharmacokinetic study to investigate its ability to cross the BBB. RESULTS: Antibodies specifically recognizing extracellular parts of ITM2A or tags inserted in its extracellular domain showed selective binding and uptake in ITM2A-overexpressing cells. However, despite high RNA expression in mouse and human microvessels, the ITM2A protein was rapidly downregulated when endothelial cells were grown in culture, probably explaining why transcytosis could not be observed in vitro. An attempt to directly demonstrate in vivo transcytosis in mice was inconclusive, using either a cross-reactive anti-ITM2A antibody or in vivo phage panning of an anti-ITM2A phage library. CONCLUSIONS: The present work describes our efforts to explore the potential of ITM2A as a target mediating transcytosis through the BBB, and highlights the multiple challenges linked to the identification of new brain delivery targets. Our data provide evidence that antibodies against ITM2A are internalized in ITM2A-overexpressing HEK293 cells, and that ITM2A is expressed in brain microvessels, but further investigations will be needed to demonstrate that ITM2A is a potential target for brain delivery.

Deciphering the immune microenvironment of a tissue by digital imaging and cognition network.

Evidence has highlighted the importance of immune cells in various gut disorders. Both the quantification and localization of these cells are essential to the understanding of the complex mechanisms implicated in these pathologies. Even if quantification can be assessed (e.g., by flow cytometry), simultaneous cell localization and quantification of whole tissues remains technically challenging. Here, we describe the use of a computer learning-based algorithm created in the Tissue Studio interface that allows for a semi-automated, robust and rapid quantitative analysis of immunofluorescence staining on whole colon sections according to their distribution in different tissue areas. Indeed, this algorithm was validated to characterize gut immune microenvironment. Its application to the preclinical colon cancer APCMin/+ mouse model is illustrated by the simultaneous counting of total leucocytes and T cell subpopulations, in the colonic mucosa, lymphoid follicles and tumors. Moreover, we quantify T cells in lymphoid follicles for which quantification is not possible with classical methods. Thus, this algorithm is a new and robust preclinical research tool, for investigating immune contexture exemplified by T cells but it is also applicable to other immune cells such as other myeloid and lymphoid populations or other cellular phenomenon along mouse gut.

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors.

KEY MESSAGE: A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay. The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora ß-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.

[Evaluation of the clinical and radiological results of cervical longitudinal median somatotomy without graft]. / Evaluation des résultats cliniques et morphologiques de la somatotomie cervicale médiane longitudinale sans greffe.

OBJECTIVES: To evaluate the clinical and radiological results of cervical longitudinal median somatotomy without graft, used for the treatment of cervical myelopathy and radiculopathy, and compare it to techniques with graft and to laminectomies. MATERIAL AND METHOD: Thirty-four patients (25 males and nine females), with a mean age over 60 years, were included in a study comparing pre- and postoperative clinical status on the Japanese Orthopaedic Association (JOA) functional scale and radiological status with evaluation of the cervical curve on plain films and dynamic tests in flexion and extension. RESULTS: No significant difference was found with the clinical and anatomical results published in the literature concerning median somatotomies performed with graft and/or osteosynthesis and laminectomies and their variants. CONCLUSIONS: The cervical longitudinal median somatotomy without graft is an easy and reliable technique that can be proposed as first-line treatment for cervical spondylotic myelopathy related to anterior compression. It decreases the cost and the duration of the surgical procedure, it protects the patient from the complications and sequelae related to graft harvesting and the use of implants. It should be limited to patients without preoperative kyphosis who are over 50 years old.

[Non traumatic spinal epidural hematomas. Report of twenty cases. Review of the literature and outcome aspects]. / Hématomes épiduraux non traumatiques du rachis. Présentation de 20 cas. Revue de la littérature et étude des aspects évolutifs.

BACKGROUND AND PURPOSE: We report a personal series of 20 non traumatic spinal epidural hematomas and study outcome aspects with a review of data in the literature. METHOD: Clinical presentation of non-traumatic spinal epidural hematomas observed between January 1980 and December 1998 was acute in 17 cases (85%) and chronic in 3 (15%). Symptoms were spinal and/or radicular pain, sensorimotor and sphincter dysfunction. Radiological evaluation consisted in myelography (n=6), myelography-CT scan (n=5), CT scan (n=1) and MRI (n=9). Patients underwent surgery in 15 cases, between 8 hours and 2 months after the first symptoms. All our patients were clinically reevaluated between 2 and 4 months after either surgery or admission for cases of spontaneous resolution. RESULTS: Good results (complete neurological resolution or moderate sequelae) were observed in 14 patients (70%). A partial recovery with major persistent neurological impairment was observed in 1 patient (5%), an initial persistent neurological impairment in 1 (5%). Three patients (15%) died and 1 (5%) was lost to follow-up. Complete spontaneous resolution were observed in four patients. CONCLUSION: Postsurgical outcome is mainly related to the preoperative neurological impairment, the duration of spinal cord compression and the time interval between the onset of symptoms and maximal deficit. A prompt laminectomy is necessary except in the cases where a spontaneous resolution can be expected from the early neurological course.

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.

Molecular characterization of CLPT1, a SEC4-like Rab/GTPase of the phytopathogenic fungus Colletotrichum lindemuthianum which is regulated by the carbon source.

The gene CLPT1 (Colletotrichum lindemuthianum Protein Transport 1) encoding a Rab/GTPase was isolated from the filamentous fungus Colletotrichum lindemuthianum, the causal agent of bean anthracnose. At the amino acid level, CLPT1 shows between 54 and 80% identity to SEC4-like proteins, a class of molecules required for intracellular vesicular transport in yeasts. In particular, typical SEC4 domains involved in nucleotide binding and membrane attachment are present in the CLPT1 sequence. Functional identity of CLPT1 with SEC4 was confirmed by complementation of the Saccharomyces cerevisiae sec4-8 mutation. This is the first report of a gene involved in the control of intracellular vesicular trafficking in a phytopathogenic fungus. RNA blot analyses of CLPT1 expression were performed during in vitro growth of the fungus on synthetic media containing glucose or pectin, as single carbon source. The accumulation of CLPT1 mRNA was strongly increased on pectin, a plant cell wall polysaccharide that induces the production of extracellular pectinases, whereas the level of CLPT1 mRNA was below the detection threshold on glucose. These results suggest that CLPT1 is mainly involved in protein secretion and that the production of extracellular enzymes potentially involved in pathogenesis in filamentous fungi is sustained by induction of the genes involved in the secretory machinery.

[Isolated facial palsy and occult adenoid cystic carcinoma of the parotid]. / Paralysie faciale isolée et carcinome adénoïde kystique occulte de la parotide.

We report a case of adenoid cystic carcinoma of the parotid disclosed by facial palsy alone. No tumefaction could be detected clinically or at imaging. The diagnosis was established at surgical exploration of the facial nerve. Total extended parotidectomy was completed by radiotherapy of the tumor site.

Construction and characterization of a catalytic fusion protein system: P-450(11beta)-adrenodoxin reductase-adrenodoxin.

Cortisol is an important intermediate for the production of steroidal drugs and can only be synthesized chemically by rather complicated multi-step procedures. The most critical step is the 11beta-hydroxylation of 11-deoxycortisol, which is catalyzed by a mitochondrial enzyme, P-450(11beta). Various fusion constructs of P-450(11beta) with its electron transfer components, adrenodoxin and adrenodoxin reductase, were produced by cDNA manipulation and were successfully expressed in COS-1 cells from which the hydroxylation activities were assayed. It was demonstrated that the fusion protein required both adrenodoxin reductase and adrenodoxin for its activity and was not able to receive electrons from an external source. The fusion protein with all three components had less activity than P-450(11beta) alone, receiving electrons from coexpressed or internal electron transfer components. The activities of the fusion proteins were determined mainly by the fusion sequence. The fusion protein with a sequence of P-450(11beta)-adrenodoxin reductase-adrenodoxin was more active than that of P-450(11beta)-adrenodoxin-adrenodoxin reductase, 1.5- and 3-fold for bovine and human P-450(11beta), respectively. Modification of the linker region by extending the size of the linker with various peptide sequences in the bovine P-450(11beta)-adrenodoxin reductase-adrenodoxin fusion protein indicated that the linker did not have significant effect on the P-450 activity. Taken together, the fusion protein obtained here can serve as a model for the investigation of electron transfer in P-450 systems and is of potential importance for biotechnological steroid production.

Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection.

The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.

Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast. Implication in in vitro cytochrome p45011beta monooxygenase system.

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made in Escherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11beta-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. Apparent Km values were determined to be 0.5, 0.6, and 0.1 microM for NADPH, NADH, and bovine adrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.

Biochemical characterization of bovine alpha s1-casein F and genotyping with sequence-specific primers.

Bovine alpha s1-casein F (alpha s1-CN F) was found in a genetic resource of Deutsches Schwarzbuntes Niederungsrind cows at a frequency of 0.009. Biochemical characterization of this new variant was obtained by automated sequencing of reversed-phase HPLC-separated tryptic peptides of alpha s1-CN F and alpha s1-CN B. alpha s1-CN F was found to be a subtype of alpha s1-CN B with a single amino acid substitution (SerP/Leu) in position 66. DNA sequencing revealed a C/T transition in position 8418 of the gene. Sequence-specific primers were designed to perform an allele-specific polymerase chain reaction for detection of alpha s1 CnF. Typing of artificial insemination sperm samples included in the genetic resource sperm pool identified one sire heterozygous for alpha s1 CnF.

Albumin standards and the measurement of serum albumin with bromcresol green. 1971.

A rapid and reliable method for measuring serum albumin employing bromcresol green is described. The addition of albumin to a solution of bromcresol green in a 0.075 M succinate buffer pH 4.20 results in an increase in absorbance at 628 nm. The absorbance-concentration relationship is linear for samples containing up to 6 g/dl albumin. Bilirubin, moderate lipemia, and salicylate do not interfere with the analysis. The use of nonionic surfactant (Brij-35) reduces the absorbance of the blank, prevents turbidity and provides linearity. The results by this method agree very well with those obtained by electrophoresis and salt fractionation. The method is simple, it has excellent precision and the reagents are stable. A protein standard is introduced which can be employed for both the total serum proteins and albumin determinations.

A gene encoding a yeast equivalent of mammalian NADPH-adrenodoxin oxidoreductases.

Adrenodoxin oxidoreductase (ADR) and adrenodoxin (ADX) are the two proteins involved in electron transport to mammalian mitochondrial P-450s capable of steroid modifications. The cloning and sequencing of a S. cervisiae ADR homologue (YADR) is presented here. The YADR protein sequence shares 36 and 37% of identical amino acids with human and bovine ADR respectively. The physiological role of this ADR homologue in yeast is unknown. We intend to study the interaction of this YADR with bovine ADX in vitro and in vivo.

11 beta-hydroxylase activity in recombinant yeast mitochondria. In vivo conversion of 11-deoxycortisol to hydrocortisone.

In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.

Isolation and sequence analysis of Clpg1, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum.

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race beta and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed on ORF encoding a 363-amino-acid (aa) polypeptide beginning with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5' non-coding region. This genomic clone was thereafter designated Clpg1. Southern analysis, performed with a Clpg1-specific probe, showed that this gene is present as a single copy in the Cl genome.

Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

New data on the proteins of rabbit (Oryctolagus cuniculus) milk.

The main rabbit milk proteins have previously been prepared by reversed-phase HPLC of the acid-precipitated material ('whole casein') and of its supernatant (acid whey). Most of them were nearly homogeneous on SDS-PAGE. Among those isolated from whole casein, alpha s1-, beta- and kappa-caseins, as well as whey acidic protein (WAP) were identified by N-terminal sequencing. After further internal sequencing, two unknown proteins were found to be the putative products, alpha s2a- and alpha s2b-caseins of two recently sequenced transcripts from rabbit mammary gland. Each whole casein component gave several bands on IEF. For kappa-casein, this was probably due to uneven glycosylation as in all kappa-caseins studied so far. For the other whole casein components, including WAP, the number of bands roughly reflected the number of potential phosphorylation sites predicted from the sequences. For alpha s1- and alpha s2-caseins polymorphism could be detected. From acid whey, in addition to WAP, which was a minor component, reversed phase HPLC separated three proteins. These were alpha-lactalbumin, transferrin and serum albumin, on the basis of their apparent molecular weights deduced from SDS-PAGE. WAP was a major component of the native whey obtained by ultracentrifugation of rabbit milk. It was found to consist of two identical subunits linked by at least one disulfide bridge.

In vivo cloning by homologous recombination in yeast using a two-plasmid-based system.

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique. Two sets of vectors were developed. The first set, called 'expression vectors', contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a 'transfer vector' which is compatible with the second set of E. coli-yeast shuttle vectors. This set of 'recombination vectors' contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 microns replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis.